GSM6932803: HL-60 RNA, DMSO, 72 hours, replicate 1; Homo sapiens; OTHER
At 72 hours, approximately 10x10^6 cells were lysed in ice cold polysome buffer (20 mM Tris pH 7.6, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL cycloheximide) supplemented with 1% v/v Triton X-100 and 25 U/mL Turbo DNase (Invitrogen). The lysates were triturated through a 27G needle and cleared for 10 min at 21,000 g at 4°C. The RNA concentrations in the lysates were determined with the Qubit RNA HS kit (Thermo). Lysate corresponding to 15 µg RNA was diluted to 200 µl in polysome buffer and digested with 0.75 µl RNaseI (Epicentre) for 45 min at room temperature. The RNaseI was then quenched by 5 µl SUPERaseIN (Thermo).     Monosomes were isolated using MicroSpin S-400 HR (Cytiva) columns, pre-equilibrated with 3 mL polysome buffer per column. 100 µl digested lysate was loaded per column (two columns were used per 200 µl sample) and centrifuged 2 min at 600 g. The RNA from the flow through was isolated using the RNA Clean and Concentrator-25 kit (Zymo). In parallel, total RNA from undigested lysates were isolated using the same kit.    Ribosome protected footprints (RPFs) were gel-purified from 15% TBE-Urea gels as 17-35 nt fragments. RPFs were then end-repaired using T4 PNK (New England Biosciences) and pre-adenylated barcoded linkers were ligated to the RPFs using T4 Rnl2(tr) K227Q (New England Biosciences). Unligated linkers were removed from the reaction by yeast 5'-deadenylase (New England Biosciences) and RecJ nuclease (New England Biosciences) treatment. RPFs ligated to barcoded linkers were pooled, and rRNA-depletion was performed using riboPOOLs (siTOOLs) per the manufacturer's recommendations. Linker-ligated RPFs were reverse transcribed with ProtoScript II RT (New England Biosciences) and gel-purified from 15% TBE-Urea gels. Ribosome protected footprints (RPFs) were gel-purified from 15% TBE-Urea gels as 17-35 nt fragments. RPFs were then end-repaired using T4 PNK (New England Biosciences) and pre-adenylated barcoded linkers were ligated to the RPFs using T4 Rnl2(tr) K227Q (New England Biosciences). Unligated linkers were removed from the reaction by yeast 5'-deadenylase (New England Biosciences) and RecJ nuclease (New England Biosciences) treatment. RPFs ligated to barcoded linkers were pooled, and rRNA-depletion was performed using riboPOOLs (siTOOLs) per the manufacturer's recommendations. Linker-ligated RPFs were reverse transcribed with ProtoScript II RT (New England Biosciences) and gel-purified from 15% TBE-Urea gels. cDNA was then circularized with CircLigase II (Epicentre) and used for library PCR. First, a small-scale library PCR was run supplemented with 1X SYBR Green and 1X ROX (Thermo) in a qPCR instrument. Then, a larger scale library PCR was run in a conventional PCR instrument, performing a number of cycles that resulted in ½ maximum signal intensity during qPCR. Library PCR was gel-purified from 8% TBE gels and sequenced on a SE50 run on an Illumina HiSeq4000.cDNA was then circularized with CircLigase II (Epicentre) and used for library PCR. First, a small-scale library PCR was run supplemented with 1X SYBR Green and 1X ROX (Thermo) in a qPCR instrument. Then, a larger scale library PCR was run in a conventional PCR instrument, performing a number of cycles that resulted in ½ maximum signal intensity during qPCR. Library PCR was gel-purified from 8% TBE gels and sequenced on a SE50 run on an Illumina HiSeq4000.